Chemical Proteomics

Volume 803 of the series Methods in Molecular Biology pp 77-95


Biotinylated Probes for the Analysis of Protein Modification by Electrophiles

  • Simona G. CodreanuAffiliated withVanderbilt University School of Medicine
  • , Hye-Young H. KimAffiliated withVanderbilt University School of Medicine
  • , Ned A. PorterAffiliated withVanderbilt University School of Medicine
  • , Daniel C. LieblerAffiliated withVanderbilt University School of Medicine Email author 

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Formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu+-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.

Key words

Electrophile Click chemistry Protein adducts Lipid oxidation Photocleavable Biotin Shotgun proteomics